The seven transmembrane receptors (also known as G protein-coupled receptors or 7TM G protein-coupled receptors) comprise a superfamily of structurally related integral proteins. 7TM G protein-coupled receptors exhibit detectable amino acid sequence similarity and all appear to share a number of structural features (See, FIG. 1). These features include: an extracellular amino terminus (EAT); seven predominantly hydrophobic alpha-helical domains (of about 20–30 amino acids) which are believed to span the cell membranes and are referred to as transmembrane domains (TMD1–7); six loops which connect the transmembrane domains (three extracellular loops (ELs) and three intracellular loops (ILs)); and a cytoplasmic carboxy terminus (CCT).
Each 7TM G protein-coupled receptor is predicted to associate with a particular heterotrimeric G protein (composed of α, β and γ subunits) at the intracellular surface of the plasma membrane. Upon binding of an agonist to the receptor, a conformational change occurs in the receptor, which enables interaction of the intracellular loops of the receptor with its associated intracellular, membrane-anchored heterotrimeric G protein. This causes the alpha-subunit of the G protein to exchange a bound GDP molecule for a GTP molecule and to dissociate from the β and γ subunits. The GTP-bound form of the alpha-subunit in turn stimulates specific intracellular signal-transducing enzymes and channels.
It has been proposed that 7TM G protein-coupled receptors adopt two major conformations: an active, G protein-coupled and thus transducing conformation and an inactive (non-transducing) conformation (Schwartz, T. W. et al., Cur. Pharmaceut. Design, 1:325–342 (1995)). The binding of an agonist or antagonist selectively stabilizes the active and the inactive receptor conformations, respectively, as predicted by the allosteric regulation of proteins as suggested by Monod, Wymann and Changeux (J. Mol. Biol., 263:7439–7442 (1965)). Agonists are thus extracellularly acting allosteric ligands that increase the signal transduction rate at intracellular sites upon binding. Antagonists are extracellularly acting ligands that inhibit signal transduction upon binding.
The 7TM G protein-coupled receptors are the largest family of cell-surface receptors comprising several hundred distinct receptors, and over 100 receptors have been cloned. The transmembrane segments of 7TM G protein-coupled receptor family members exhibit considerable homology, whereas the extracellular connecting loops are less conserved, showing high homology only between closely related receptor subtypes. The 7TM G protein-coupled receptors can be grouped based on their homology levels and/or the nature of the ligands they recognize. For example, the interleukin-8 receptor, the angiotensin II receptor, the thrombin receptor, the endothelin receptors, the N-formyl peptide receptor and the C5a receptor all bind peptide ligands and share 20–40% amino acid similarity.
The 7TM G protein-coupled receptors bind a wide variety of ligands of different molecular size ranging from small monoamines and other small molecules, to large neurotransmitters and peptide hormones. The family of 7TM G protein-coupled receptors also includes the receptors for light (rhodopsin), for odors (olfactory receptors) and for taste (gustatory receptors). Additionally, the conserved structure among 7TM G protein-coupled receptors has allowed for the cloning of many novel genes encoding 7TM G protein-coupled receptors whose natural ligand and function are yet to be elucidated. These receptors are referred to as “orphan” receptors. Table 1 lists a number of 7TM G protein-coupled receptors which have been cloned and expressed.
Because of the involvement of 7TM G-protein-coupled receptors in the regulation of many critically important biological functions and disease conditions, many of these functions and conditions may be influenced or determined by the state of activation or inhibition (e.g., blockade) of a 7TM G protein-coupled receptor. However, these receptors are difficult to purify. The proteins can be removed from the membrane only by the action of detergents, which denatures some proteins. In addition, most membrane proteins are not soluble in water. To date, few novel agonists or antagonists to these receptors have been identified. Common methods have involved generating antibodies to 7TM G protein-coupled receptors expressed in cells which have been administered to a host. Lerner et al. (PCT application No. WO 98/03632) have described peptide dimer agonists for 7TM G protein-coupled receptors. These dimers were comprised of two known peptide agonists or antagonists (e.g., natural ligands) to different 7TM G protein-coupled receptors.
It would be useful to be able to develop agonists and antagonists to the specific binding portions of 7TM G protein-coupled receptors. Attempts to achieve expression of only the ligand binding portion of a 7TM G protein-coupled receptor have been unreproducible or have resulted in inefficient and/or unpredictable levels of expression (Xie, U. B., et al, J. Biol. Chem. 265:21441–21420 (1990); Tsai-Morris, C. H., et al. J. Biol. Chem. 265:19385–19388 (1990)).
As suggested in Lerner et al., bivalent binding molecules can have utility as therapeutics. More specifically, bivalent and bispecific antibodies have many practical applications, including in immunodiagnosis and therapy. Bivalency can allow antibodies to bind to multimeric antigens with great avidity; multivalency theoretically can increase apparent binding affinity by several orders of magnitude (Crothers, D. M. et al., Immunochemistry 9: 341–351 (1972)). Bispecificity can allow the cross-linking of two antigens, for example, in recruiting cytotoxic T cells to mediate killing of a tumor cell. Specific examples of bivalent molecules capable of binding to adjacent epitopes include small bivalent antibodies composed of either antibody fragments (Fab) or single chain antibodies (Fv) (Pack, P. et al., Biochemistry 31, 1579–1584 (1992); Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90, 6444–6448 (1993); Mallender, W. D. et al., J. Biol. Chem., 269: 199–206 (1994)). Neri, D. et al. (J. Mol. Biol., 246:367–373 (1995)) developed a bispecific antibody fragment, binding two antibodies with a polypeptide chain, that recognizes adjacent and non-overlapping epitopes of lysozyme and is able to bind both epitopes simultaneously.
Bivalent peptides, such as receptor-adhesive modular proteins (“RAMPs”), have been used in an alternative approach to cell targeting. M. Engel et al., (Biochemistry 30: 3161–3169 (1991)) and C. A. Slate et al., (Int. J. Peptide Protein Res. 45: 290–298 (1995)) have designed large synthetic peptides, which contain two ligand sites separated by a spacer region and a dimerization domain.
The SELEX Process
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential Enrichment, termed the SELEX process, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned; U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which is herein specifically incorporated by reference. These applications, collectively referred to herein as the SELEX patent applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on the Basis of Structure”, abandoned in favor of U.S. Ser. No. 08/198,670, now U.S. Pat. No. 5,707,796, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands”, abandoned in favor of U.S. Ser. No. 08/443,959, filed May 18, 1995, which was abandoned in favor of U.S. Ser. No. 08/612,895, filed Sep. 16, 1994, now U.S. Pat. No. 5,763,177, describes a SELEX-based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine”, now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX”, abandoned in favor of U.S. Ser. No. 08/461,069, filed Jun. 5, 1995, now U.S. Pat. No. 5,567,588, and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled “Flow Cell SELEX”, now U.S. Pat. No. 5,861,254, describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev”, now U.S. Pat. No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX”, now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides”, abandoned in favor of U.S. Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, now U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement”, now U.S. Pat. No. 5,756,703, describes oligonucleotides containing various 2′-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX”, now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX”, now U.S. Pat. No. 5,683,867, respectively. The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic or Non-Immunogenic, High Molecular Weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid Ligand Complexes”, now U.S. Pat. No. 6,011,020. VEGF Nucleic Acid Ligands that are associated with a Lipophilic Compound, such as diacyl glycerol or dialkyl glycerol, in a diagnostic or therapeutic complex are described in U.S. patent application Ser. No. 08/739,109, filed Oct. 25, 1996, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes”, now U.S. Pat. No. 5,859,228. VEGF Nucleic Acid Ligands that are associated with a Lipophilic Compound, such as a glycerol lipid, or a Non-Immunogenic, High Molecular Weight Compound, such as polyalkylene glycol, are further described in U.S. patent application Ser. No. 08/897,351, filed Jul. 21, 1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes”. VEGF Nucleic Acid Ligands that are associated with a non-immunogenic, high molecular weight compound or lipophilic compound are also further described in PCT application Publication No. WO 98/18480, filed Oct. 17, 1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes”. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
The identification of nucleic acid ligands to small, flexible peptides via the SELEX method has been explored. Small peptides have flexible structures and usually exist in solution as an equilibrium of multiple conformers, and thus it was initially thought that binding affinities may be limited by the conformational entropy lost upon binding a flexible peptide. However, the feasibility of identifying nucleic acid ligands to small peptides in solution was demonstrated in U.S. Pat. No. 5,648,214, filed Sep. 9, 1994, entitled “High-Affinity Oligonucleotide Ligands to the Tachykinin Substance P”, which is incorporated herein by reference. In this patent, high affinity RNA nucleic acid ligands to substance P, an 11 amino acid peptide were identified.
Xu and Ellington (Proc. Natl. Acad. Sci. USA, 93:7475–7480 (1996)) employed the human immunodeficiency virus type 1 (HIV-1) Rev to further explore how peptide and protein epitopes are recognized by nucleic acid ligands. In this study, RNA nucleic acid ligands were selected to bind to the isolated Rev34–50 peptide. It was observed that RNA nucleic acid ligands could not only recognize the sequence of this peptide, but that these nucleic acid ligands could also bind the corresponding native epitope on the Rev protein, albeit with lower affinity.
The present invention provides bivalent binding molecules comprising two or more binding domains which bind simultaneously to two or more epitopes of the same 7TM G protein-coupled receptor and thus increase the binding affinity relative to the binding of a single binding domain. The binding domains are identified using synthetic peptides corresponding to all or a portion of the extracellular binding domains and therefore purified and isolated receptor proteins are not required.